Functional organization of the CCAN protein complex

Kinetochores, huge proteinaceous scaffolds, assemble on centromeric chromatin and bind spindle microtubules to faithfully segregate sister chromatids during mitosis. The centromere - kinetochore interface is build by the constitutive centromere associated network (CCAN). CENP-C, a multi-functional CCAN protein, has been proposed to be one of the key players of kinetochore assembly. CENP-C is known to bind the outer kinetochore component Mis12 complex with its very N-terminus, its central domain is interacting with CENP-A nucleosomes, whereas its C-terminus has been suggested to be involved in CENP-A deposition. Although, dependency of several CCAN components on CENP-C for their kinetochore localization has been demonstrated, CENP-Cs role within CCAN assembly remains insufficiently understood. This study provides insights into organization of CENP-C within the CCAN, both by biochemical and structural approaches using recombinant material, but also within the context of human cells. It is clearly demonstrated that the N-terminal half of CENP-C is the assembly platform of the CCAN by directly recruiting the CENP-H/I/K/M and CENP-N/L complexes onto a region of CENP-C located between the Mis12 complex and the CENP-A binding sites. Point mutations within conserved patches of this CENP-C motif specfiically destroyed the CENP-C:CENP-H/I/K/M interaction, which was followed by a loss of CENP-N/L and CENP-T/W within human cells. CENP-T/W has proven to directly bind CENP-H/I/K/M in a previous study. A direct interaction of CENP-T/W with CENP-C was not observed. Taken together, CENP-C directly binds to CENP-H/I/K/M, that together provide a platform for CENP-N/L assembly. CENP-T/W indirectly depends on CENP-C through its interaction with the CENP-H/I/K/M complex. Therefore, CENP-C represents the only CCAN assembly interface within the kinetochore and thus displays a crucial component for kinetochore constitution.

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